Plates were coated with 1 g of Stx2 (Phoenix Lab, Tufts University or college, Boston, MA). encoding the 14 amino acid Dansylamide albumin-binding-peptide (ABP), DICLPRWGCLEWED [20] is usually fused in frame to the carboxyl end of the H7/B5 coding DNA, separated by a GGGGS spacer. Two copies of the E-tag peptide epitope are also encoded (E), one at the amino end of ciA-H7 and the other between ciA-B5 and ABP, and utilized for detection in this study. Boxes are not to level. (B) Amino acid sequence of the encoded VNA-BoNTA. The complete amino acid sequence of the encoded VNA-BoNTA within Ad/VNA-BoNTA as diagrammed in A is shown. For clarity, the eight different protein domains shown in A are separated by///. (C) Amino acid sequence of VNA-Stx (VNA-A9/A5/G1/ABP) encoded by Ad/VNA-Stx. The complete amino acid sequence VNA-Stx [6] within Ad/VNA-Stx is shown. For clarity, the different protein domains (SP; three Stx-neutralizing VHHs Stx1-A9, Stx-A5, Stx2-G1 [6]; E; ABP) are separated by///as in B.(TIF) pone.0106422.s002.tif (328K) GUID:?71168FFB-0E9A-41FD-8E6B-31CD9F719768 Figure S3: Serum VNA titers following Ad/VNA treatments via different routes of administration. Dilution ELISAs were performed to assess the VNA-BoNTA (A) or VNA-Stx (B) titers from five individual mice (solid lines) four days following treatment with 31010 vp of Ad/VNA-BoNA or Ad/VNA-Stx respectively. Treatments were administered via intravenous, intraperitoneal, intramuscular or subcutaneous routes (individual boxes). ELISA plates were coated with 1 g/ml of ciBoNT/A (A) or Stx2 (B). The first well contained a 110 dilution of serum followed by a 15 dilution series. A control mouse serum (dashed lines) was spiked with 400 nM of recombinant VNA-BoNTA (Trx/H7/B5/ABP) (A), or VNA-Stx (Trx/A9/A5/G1/ABP) Dansylamide (B). VNA binding to toxin was detected with HRP/anti-E-tag (vertical axis). The horizontal axis label shows the concentration of the Trx/VNA standard.(TIFF) pone.0106422.s003.tiff (3.1M) GUID:?1C63C550-E1DC-454C-9BCB-9AA0BB5BD485 Data Availability StatementThe authors confirm Rabbit polyclonal to FANK1 that all data underlying the findings are fully available without restriction. All relevant data are included within the paper and its Supporting Information files. Abstract Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing brokers (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only VH domains (VHHs), each binding unique epitopes, can potently safeguard animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 1 to 2 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was designed that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous Dansylamide > intraperitoneal > Dansylamide intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% guarded from 10 LD50 of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully guarded from 10 LD50 BoNT/A1 when Ad/VNA-BoNTA was administered up to 1 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases. Introduction Botulism is usually a flaccid paralysis caused by exposure to Botulinum neurotoxin (BoNT) that results primarily from ingestion of contaminated foods, although the risk of exposure through deliberate events is considered sufficiently high to list BoNT as a Category A Priority Pathogen. Toxin exposure is commonly treated by administration of antitoxin serum, generally prepared from large animals immunized with inactivated toxin [1]C[3]. Such antiserum products possess safety risks and are hard to develop, produce and maintain. Antiserum is also not practical for prophylactic protection of people that are considered at-risk of toxin exposures. BoNT antiserum alternatives, such as monoclonal antibodies (mAbs) are under development [4] and other strategies are in the research stage [1]. We have reported the use of an alternative antitoxin strategy [5] which employs VHH-based neutralizing brokers (VNAs) consisting of linked 14 kDa camelid heavy-chain-only VH domains (VHHs), produced as heteromultimers, that bind and neutralize toxin targets. VNAs were found.